This is a competitive renewal of an R24 grant focused on mitochondrial protein acylation, its regulation by the NAD-dependent mitochondrial protein deacylases, SIRT3 and SIRT5, and their impact on metabolic regulation under normal and pathological conditions. During the first 48 months of this grant, our collaborative group has published 27 papers and has defined the function of SIRT3 and SIRT5 as key mitochondrial NAD-dependent protein deacylases. In the mitochondria, SIRT3 and SIRT5 regulates the acetylation (SIRT3) and succinylation (SIRT5) of many mitochondrial enzymes in key metabolic pathways that include fatty acid oxidation, ketone body synthesis, TCA cycle, OXPHOS and the urea cycle. Loss of SIRT3 is associated with reduced metabolic flexibility and accelerated development of a syndrome that mimics human metabolic syndrome with obesity, type 2 diabetes, lipid abnormalities, and steatohepatitis. Mice lacking SIRT5 also showed significant metabolic abnormalities in the urea cycle, ketogenesis and fatty acid oxidation. SIRT5 is also an NAD-dependent demalonylase but this modification targets distinct proteins predominantly in the cytoplasm. These findings point to reversible mitochondrial protein acylation as a key regulator of mitochondrial metabolism and SIRT3 and SIRT5 as important factors in the pathogenesis of type 2 diabetes and the metabolic syndrome. The overall goal of this proposal is to extend these studies to further define the role of protein acylation and SIRT3 and SIRT5 in mitochondrial function in unique tissues involved in the pathogenesis of the metabolic syndrome: liver, muscle and pancreatic cells. We will take advantage of our highly collaborative and multidisciplinary team harnessing the power of mass spectrometry-based proteomics, metabolomics, molecular biology, extensive physiological testing and unique and novel animal models, including tissue-specific knockouts and tissue specific transgenic overexpressors for SIRT3 and SIRT5, to further increase our understanding of this important process in regulation of mitochondrial function and metabolism.